The vascular permeability factor (PF) of Pseudomonas aeruginosa in culture liquids was purified by the following steps: removal of nucleic acid with manganese chloride, filtration through 450-nm membranes (Millipore), concentration with PM30 ultrafiltration membranes (Amicon), ammonium sulfate fractionation, gel filtration on Sephadex G- 200 column, and DEAE cellulose column chromatography. Analytical polyacrylamide gel electrophoresis revealed one major band and a few minor bands. The purification process resulted in a great loss of PF activity and qualitative changes in skin lesions in rabbits. Fractions in the void volume from a Sephadex G-200 column, to which a concentrated culture filtrate was applied, were pooled and rechromatographed through a similar Sephadex G-200 column, and all the fractions in the void volume were pooled. When this pool, which by itself had no PF activity, was added to the purified PF, the original lesion-producing capacity was restored. The active substance in this pool, which we designate as cofactor, is resistant to heat and trypsin and is effective in quantities as little as 0.1 microgram per skin spot. A lipopolysaccharide preparation made with the same strain as used for PF production by the phenol method of Westphal and Jann exhibited cofactor activity.